Schistosoma mansoni: incorporation and metabolism of protein amino acids in vitro

1. The amino acid composition of total proteins in six stages of the life cycle of Schistosoma mansoni was determined by routine autoanalysis of acid hydrolysates. Aspartate, glutamate and glycine were consistently the most abundant protein amino acids in all stages. 2. Incorporation of each of the protein amino acids into adult and egg proteins was determined using 72 hr cultures in complex media. Incorporation rates varied widely and there was no correlation between abundance in protein and the rate of incorporation. 3. Only five amino acids were interconverted to other amino acids which were themselves incorporated into worm and egg proteins (ala, arg, asp, gly, ser); of these only two (glu from ala and pro from arg) appeared to be of quantitative significance. Exogenous glucose yielded only three protein amino acids (ala, asp, glu). 4. The data are considered in the light of differences in egg and adult protein synthesis and with particular regard to potential chemotherapy at this level.     

New Mycotoxin, Trichotoxin A, from Trichoderma viride Isolated from Southern Leaf Blight-Infected Corn

A new mycotoxin, trichotoxin A, was found in a solvent extract of the mycelium of Trichoderma viride isolated from corn infected with southern leaf blight. Trichotoxin A is a cyclic peptide with the following amino acid composition: (gluN)(2), (glu)(1), (pro)(2), (gly)(1), (ala)(3), (leu)(3), (2-methyl alanine)(1).     

Evidence for covalent binding between phosphopeptides and terminal deoxynucleotides in highly purified calf thymus DNA

Summary Highly purified DNA from calf thymus nuclei (N-DNA) was found to cleave after reaction with a chelating agent and subsequent dialysis. During the cleavage phosphopeptides (PPs) were released into the dialysates. At the end of the cleavage, approximately one half of the PP material remained with the DNA. Since it was so strongly bound, it was considered to be retained in the DNA structure by covalent bonding. In order to confirm this, a commercial DNA (S-DNA) was ultrasonicated and digested with pancreatic DNAase, exonuclease III, and S1 nuclease. DEAE Sephacel chromatography of the digested material yielded 5 fractions. The fraction 2, having the highest proportion of proteinaceous material, was digested with Pronase. Amino acid analysis of the hydrolysis mixture yielded phosphoserine (Pser), asp, thr, ser, glu, gly, ala, val, ile, leu, and arg. The mixture was chromatographed again on DEAE Sephacel. From this a single fraction, number 5, was found to contain both deoxynucleotides and the amino acids, Pser, asp, ser, glu, and gly in a molar ratio of > 7:3:2:2:5. The mixture obtained by hydrolysis of this fraction with snake venom diesterase was again chromatographed on DEAE Sephacel. This fractionation gave two main peaks, one corresponding to the same 5 amino acids and the other to deoxynucleotide material. From this it was concluded that the fraction used for diesterase digestion consisted of deoxynucleotide-amino acids, with covalent diester bonds between the deoxynucleotide and amino acid portions.Dedicated to Prof. L.E. Feinendegen on the occasion of his 60th birthday     

THE IN VITRO FORMATION OF ACETYL-ASPARTYL-PEPTIDO-SEROTONIN COMPLEXES BY PIG HYPOTHALAMIC TISSUE EXTRACTS

Peptido-serotonin complexes are formed on incubation of pig hypothalamic tissue extracts with acetyl-aspartate, the amino acids glu. ser, gly and ala. an ATP-regenerating system and serotonin. The purification and partial characterisation of two acetyl-aspartyl-peptido-serotonin complexes is described.     

Excess of taurine supplementation in rat: effects on GABA-related amino acids in developing nervous tissues.

The effects of taurine supplementation on GABA-related amino acid homeostasis in developing nervous tissues of suckling rats were studied. In the first two weeks of postnatal growth, cerebral cortex and cerebellum appear more accessible to taurine supplementation in comparison to retina; in addition, different changes in excitatory/inhibitory amino acids were observed. After the 5th day of life, in the retina and cerebellum of taurine-supplemented pups a decrease in GABA levels was found; in contrast, in cerebral cortex GABA content significantly increased throughout 20 days of postnatal growth. In all nervous tissues studied (except for cerebellum) glutamine concentration increased at the 5th day; then in cerebellum and in retina, but not in cerebral cortex, a significant decrease until the 20th day occurred. Furthermore, in cerebellum and retina taurine supplementation decreased glutamate levels, in comparison to controls, at the 10th and until the 20th day of postnatal life, respectively, whereas in cerebral cortex an increase in glutamate level was observed only at the 5th day. In conclusion, taurine supplementation, in excess to the usual amount from the mother's milk, affected the glutamate compartments in various cell types. The changes in GABA-related amino acid concentrations in cerebral cortex, cerebellum, and retina may depend on the different pattern of the metabolic processes at different maturative stages.     

Variations circadiennes des acides aminés libres de l'hemolymphe de Penaeus japonicus

The non-essential free amino acids of Peneaus japonicus hemolymph (asp, ser, glu, pro, gly, tyr, arg, ala, cys, tau) are more common than the essential ones: 750 pmol μl^?1 of hemolymph vs 330 pmol. Under a light-dark (L-D); 12-12 photoperiod, tricircadian variations of males or tetracircadian variations of females are more pronounced for non-essential amino acids then for essential ones. In the first case, free amino acid concentrations of hemolymph can be multiplied by a factor of three, and in the second case by a factor of six; but circadian variations of females are greater than those of males. Differences between the maximum and minimum of essential free amino acid concentrations of male and female hemolymph are more frequent than for non-essential amino acids. A differences of 2 h between the minimum of essential and non-essential amino acid concentrations in males only appeared during the afternoon. The more concentrated free amino acids in P. japnicus hemolymph are glycine, proline, histidine, and alanine; the less concentrated are lysine, cysteine and glutamic acid while others, like leucine, isoleucine, phenylalanine and tyrosine can only be estimated at 10.00 and 24.00 h.     

Cell wall composition and deoxyribonucleic acid similarities among the anaerobic coryneforms, classical propionibacteria, and strains of Arachnia propionica

Eighty strains of anaerobic coryneforms were compared with 29 strains of classical propionibacteria and 8 strains of Arachnia propionica by cell wall analysis, deoxyribonucleic acid (DNA) base compositions, and nucleotide sequence similarities. The anaerobic coryneforms have DNA base compositions in the range of 58 to 64% guanine + cytosine (GC) and show at least three homology groups. The largest group corresponds to organisms identified as Propionibacterium acnes and shows about 50% homology to strains in the P. avidum homology group. The third group, P. granulosum, shows low levels of similarities to the other two. All strains of anaerobic coryneforms have some combination of galactose, glucose, or mannose as cell wall sugars, and most have alanine (ala), glutamic acid (glu), glycine (gly), and l-alpha-epsilon-diaminopimelic acid (l-DAP) as amino acids of peptidoglycan. However, a few strains in the P. acnes and P. avidum homology groups have meso-DAP and minimal amounts of glycine. Two serological types, based on cell wall antigens, were found in the P. acnes homology group. One type had galactose, glucose, and mannose as cell wall sugars, the other glucose and mannose only. The classical propionibacteria have DNA base compositions in the range of 65 to 68% GC and show four homology groups which correspond closely to van Niel's classification as given in the 7th edition of Bergey's Manual. The P. jensenii group showed about 50% homology to the P. thoenii group and about 30 to 35% to the P. acidi-propionici group. The P. freudenreichii strains showed a rather lower level of similarity (8 to 25%) to the other homology groups. Most of the strains of classical propionibacteria also have some combination of galactose, glucose, or mannose as cell wall sugars and ala, glu, gly, and l-DAP as peptidoglycan amino acids, but P. shermanii and P. freudenreichii strains, which form a single homology group, have galactose, mannose, and rhamnose as cell wall sugars and ala, glu, and meso-DAP in their peptidoglycan. There is a rather low level of DNA homology (10 to 20%) between the anaerobic coryneforms and classical propionibacteria. However, the strains of A. propionica which have a GC content of 64 to 65% and form a single homology group, show no homology to either of the other two major groups.     

FREE AMINO ACIDS IN DEVELOPING RAT RETINA

—During postnatal growth the free amino acids pattern of rat retina differs at various developmental stages. The adult level for individual amino acids is reached on the 30th day of maturation. During differentiation the taurine, glutamic acid, GABA, glutamine, aspartic acid, glycine arginine, methionine and histidine levels increase while proline. alanine, ornithine and tyrosine decrease.     

Behavioural responses of glass eels (Anguilla anguilla) towards amino acids

The behavioural responses of glass eels of Anguilla anguilla towards amino acids were investigated by binary choice experiments testing different concentrations of 14 L-amino acids in fresh water and salt water. Glass eels responded to solutions of individual amino acids down to a concentration of 10⁻⁸M, 10⁻⁹m. Media of different salinities influenced the responses. In freshwater, gln and thr were strongly attractive; asn, ala, met, glu and ile induced significant avoidance; gly elicited multimodal reactions depending on concentrations; his, lys, phe, val, leu and asp hardly influenced behaviour. In salt water, only gly, asn and lys significantly influenced the behaviour of the glass eels.     

Purification and characterization of the myoglobins of Paramecium tetraurelia.

1. The molecular variants of the myoglobins of Paramecium tetraurelia have been purified as five separate components and designated Mb 1 to Mb 5. 2. The five molecular forms are homogeneous on polyacrylamide gel electrophoresis (PAGE) and isoelectric focusing (IF). 3. The myoglobin species appear to have identical molecular weights and amino acid compositions and differ only in their isoelectric points and relative concentrations in vivo. 4. The myoglobin species have an apparent molecular weight of 15,000 +/- 500 and possess a single heme group per mole which appears to be protoporphyrin IX. 5. The amino acid composition of the five species is: lys12, his3, arg4, asp17, thr9, ser6, glu16, pro4, gly11, ala15, cys0, val8, met2, ile7, leu10, tyr5, phe7. 6. The spectra of several ferrous and ferric derivatives of the mixture Mb 1-Mb 5 are presented.     

Phosphopeptides in highly purified calf thymus DNA

Highly purified DNA obtained from calf thymus nuclei was found to cleave after reaction with a chelating agent and subsequent dialysis against 0.01 M phosphate. During the cleavage release of proteineous material into the dialysate was observed. By means of anion exchange resin column chromatography, this material was separated into 9 main fractions. Two of these fractions P1 and P5) were found to contain the amino acids phosphoserine, asp, thr, ser, glu, gly, ala, val, ile, leu, and arg, as well as metal ion complexes of phosphoserine. The complexes were dissociated by Chelex 100 treatment. The proportion of phosphoserine was much greater in P5 than in P1. P1 and P5 contained essentially no nucleotide material. All other fractions (P2, P3, P3a, P4, P5a, P6, P7, P8, P6a, P9) were found to contain ribonucleotides and deoxynucleotides. The deoxynucleotide content was about 10% of total nucleotide content. After a deionizing treatment with Chelex, the amounts of nucleotides were extensively reduced to a level corresponding to about 1 nucleotide of 10 amino acids. In separate experiments, commercial DNA (S-DNA) was ultrasonicated, and digested with pancreatic DNAase, exonuclease III, and S1 nuclease. From DEAE Sephacel chromatography of this material the fraction obtained having the highest proportion of protein aceous material was hydrolyzed with Pronase and again chromatographed on DEAE Sephacel. From this fractionation a single fraction containing deoxynucleotides and amino acids was found. The mixture obtained by hydrolysis of this fraction with snake venom diesterase and was again rechromatographed, which revealed two peaks, one corresponding to deoxynucleotide material and a second one to a mixture of 4 amino acids, phosphoserine, asp, glu, and gly. From this it was concluded that the fraction used for diesterase digestion consisted of deoxynucleotide-amino acids, with covalent diester bonds between their deoxynucleotide and amino acid portions. The results indicate that in purified S-DNA phosphopeptides are linked through covalent bonds to the terminal deoxynucleotide residues.     

Characterization of phosphopeptides released during dialysis of EDTA-reacted highly purified DNA prepared from calf thymus nuclei

The treatment of highly purified DNA obtained from calf thymus nuclei (N-DNA) with a chelating agent and subsequent repeated dialyses led to release of phosphopeptides (PPs) into the dialysates. By means of anion exchange column chromatography, the PPs were separated into 9 main fractions. Two of them (P1 and P5) contained the amino acids phosphoserine, asp, thr, ser, glu, gly, ala, val, ile, leu, and arg, as well as metal ion complexes of phosphoserine. The complexes were dissociated by deionization with nitrilotriacetate + Chelex. The proportion of phosphoserine was about twice as great in P5 as in P1. Whereas P1 and P5 contained essentially no nucleotide material, the other fractions contained ribonucleotides and deoxynucleotides. The deoxynucleotide content was less than 10% of that of total nucleotides. After a deionizing treatment, the amounts of nucleotides in these fractions were reduced to a level corresponding to 1 nucleotide per peptide of 5-15 amino acids.     

Perinatal changes in amino acid metabolism of rat brain,especially alanine and glutamic acid

The changes in both the levels of some free amino acids and their metabolism in the rat brain during the first 24 hr of postnatal life were studied. The content of glutamic acid decreased for the first 2 hr; it remained at the lowest level for the next 4 hr, when it began to increase. The content of alanine decreased for the first 6 hr and approached the adult level. Oxygen consumption, glucose oxidation, and pyruvate formation in the cerebral slices of the 24-hr-old rats were as much as 150% of that of the 19-day-old fetus. The distribution profile of radioactivity incorporated into the cerebral amino acids from the subarachnoid-injected [U¹⁴C]glucose was also changed. In the 2- and 6-hr-old rats, 50% of the total radio-activity recovered in the free amino acids was in alanine. Its rate decreased to 30% in the 24-hr-old and was 2% in the adult, while the radioactivity incorporated into glutamic acid increased. Alanine aminotransferase activity started to increase at birth and had the highest level at 24 hr after birth. It then decreased and finally reached the same level as shown at birth. However, aspartate aminotransferase increased during the first 6 hr after birth and did not change until the end of the first day of life.     

Isolation and characterization of human platelet glycoprotein IX

Human platelet glycoprotein IX is a small (Mr 17,000) surface glycoprotein present in the plasma membrane as a 1:1 non-covalent complex with glycoprotein Ib (Mr 165,000). Glycoprotein IX was purified to near homogeneity by sequential wheat germ agglutinin and immuno-affinity chromatography, followed by gel filtration chromatography under denaturing conditions. Purified glycoprotein IX was characterized by determination of its amino acid composition and its NH2-terminal amino acid sequence (thr-lys-asp-xxx-pro-ser-xxx-leu-thr-(thr)2-arg-ala-(leu)-(glu)-xxx-met- gly), and monospecific anti-glycoprotein IX antibody was prepared. The study outlines a useful approach for separating and characterizing glycoprotein IX, free from other members of the glycoprotein Ib complex.     

Silica, Alumina, and Clay-Catalyzed Alanine Peptide Bond Formation

The peptide bond formation of alanine (ala), ala + glycine (gly), ala + diglycine (gly₂), and ala + gly cyclic anhydride (cyc-gly₂) in drying/wetting cycles at 80°C was studied. Silica, alumina, and representative smectites—montmorillonite and hectorite—were used as catalysts, and the dependence of reaction yields on the available amount of water in the reaction systems was evaluated. Silica and alumina catalyze the formation of oligopeptide mainly in temperature fluctuation experiments, whereas higher amounts of water in the reaction system support clay-catalyzed reactions. Silica and alumina are much more efficient for amino acid dimerization than clays. Whereas only 0.1% of ala oligomerized on hectorite and no reaction proceeded on montmorillonite, about 0.9 and 3.8% alanine converted into its dimer and cyclic anhydride on silica and alumina, respectively. Clay minerals, on the other hand, seem to more efficiently catalyze peptide chain elongation than amino acid dimerization. The reaction yields of ala-gly-gly and gly-gly-ala from ala + gly₂ and ala + cyc-gly₂ reached about 0.3% on montmorillonite and 1.0% on hectorite. The possible mechanisms of these reactions and the relevance of the results for prebiotic chemistry are discussed. Received: 15 December 1996 / Accepted: 1 May 1997     

POSTNATAL CHANGES IN THE LEVELS OF GLYCINE AND THE ACTIVITIES OF SERINE HYDROXYMETHYLTRANSFERASE AND GLYCINE: 2-OXOGLUTARATE AMINOTRANSFERASE IN THE RAT CENTRAL NERVOUS SYSTEM

Abstract— Of the amino acids found in the CNS of 10-day-old rats the concentration of glycine alone was significantly higher in the spinal cord than in all other regions. Spinal levels of glycine, cystathionine, isoleucine and lysine from 1- and 10-day-old rats did not differ significantly from adult values, whereas the levels of most other amino acids, including GABA, glutamate, glutamine and taurine, were higher in the young animals than in the adults. Aspartate was the only amino acid found in lower concentration in the spinal cord of young animals than in adult animals. These and other observations support the conclusion that glycine is used as an inhibitory transmitter in rat spinal cord early in postnatal life. There was a general decrease in the activity of serine hydroxymethyltransferase and a slight increase in the activity of glycine:2-oxoglutarate aminotransferase in the CNS during development. The activity of neither enzyme correlated on a regional basis with the glycine content. The high level of hydroxymethyltransferase activity in the cerebellum of 10-day-old rats suggests that the activity of this enzyme reflects cell growth rate.     

The Use of Inhibitors of GABA-Transaminase for the Determination of GABA Turnover in Mouse Brain Regions: An Evaluation of Aminooxyacetic Acid and Gabaculine

Abstract: The accumulation of γ -aminobutyric acid (GABA) after inhibition of GABA-T (4-aminobutyrate: 2-oxoglutamate aminotransferase, EC 2.6.1.19) by various doses of aminooxyacetic acid (AOAA) and gabaculine was studied in four different regions of the mouse brain. The dose-response curve for GABA accumulation after treatment with AOAA was linear up to 10 mg/kg i.p., and then leveled off. The increase in GABA accumulation after gabaculine treatment was linear up to 100 mg/kg i.p. No further increase was observed with doses up to 300 mg/kg i.p. The selectivity of both GABA-T inhibitors was assessed by measuring their effects on the content of free amino acids in mouse brain. Apart from the substantial increase in the GABA concentration, there were significant decreases in the content of glutamic acid, aspartic acid, alanine and glutamine, and an increase in ornithine content after administration of gabaculine. The same changes in amino acid content were observed after treatment with AOAA, but the level of lysine was also increased and the change in alanine level was biphasic. All these changes, however, were very small compared with the large increase in GABA level. A method for estimating the rate of the GABA turnover in vivo by measuring the initial rate of GABA accumulation after administration of AOAA or gabaculine is proposed, and the validity of the two techniques is discussed. The effect of diazepam on GABA levels and on the gabaculine-induced accumulation of GABA was studied. The results obtained with diazepam show that this method can provide valuable insight into the effects of drugs on GABAergic mechanisms in vivo.     

Effect of vitamin B3-active compounds on the content of free and combined gamma-aminobutyric acid and glutamic acid in the brain of mice

The bound and free GABA and glutamic acid content in the brain of F1 (CBA X C57B1/6) hybrid mice was investigated by the Eliott method. A tendency to a decrease of GABA and glutamate content in the brain with their practically constant bound/free ratio was observed 24 h after calcium-D-pantothenate injections (150 mumole/kg, 9 injections for 3 days). Calcium-D-homopantothenate injected in the same way caused a significant decrease in the GABA content, and a sharp drop of the bound/free GABA ratio. The effect is not associated with the influence of calcium ions in the composition of the injected compounds.     

Free Amino Acid Content of Astroglial Cell Clones Derived from 8-Day Postnatal Mouse Cerebella

We have measured the free amino acid content of three distinct astroglial cell clones derived from permanent lines obtained after "spontaneous immortalization" of 8-day postnatal mouse cerebellar cultures; these clones show characteristics similar to the Golgi Bergmann glia cells, the fibrous astrocytes, and the velate protoplasmic astrocytes, i.e., the three main types of cerebellar astrocytes. The relative concentrations of amino acids that are thought to act as neurotransmitters were compared in confluent cultures of the different astroglial clones. The most striking result was a high concentration of glycine (20% of free amino acids), even in astroglial cells cultured in a glycine-free medium, a finding suggesting that glycine is synthesized by the astroglial clones. Furthermore, no gamma-aminobutyric acid (GABA) was detected. In contrast, a "neuron-like" clone derived from the same cerebellar culture contained GABA, whereas its glycine content was much lower than that of the astroglial clones. The present results, together with our previous finding of glycine synthesis in an astrocytic clone derived from 14-day postnatal mouse cerebella transformed by simian virus 40, indicate that a high glycine content may be characteristic of many cerebellar astroglial types.